Exo-endocytosis at Neuromuscular Junctions during Intense Quantal Release of Neurotransmitter

Abstract

Freeze-fracture technique was used to study frog neuromuscular junctions that had been treated with black widow spider venom, K + rich solutions or that had been stimulated indirectly. In the presynaptic membranes of nerve terminals that had been treated with venom for 8–15 min numerous dimples (E face) or protuberances (P face) were found immediately adjacent to the double row of particles that line the active zones. In contrast no dimples or protuberances were found in the nerve terminals that had been treated for 1 h and which were depleted of synaptic vesicles, Numerous dimples or protuberances were also found in the presynaptic membranes of nerve terminals that had been stimulated indirectly or treated with 20 mM K + . However, under these conditions the dimples or protuberances were not confined to the double rows of particles, but were scattered all over the presynaptic membrane. Dimples or protuberances were still found after 1 h soaking in 20 mM K + , showing that extensive recycling of synaptic vesicles had occurred. Recycling in K + rich solutions was confirmed by thin section EM and by experiments with extracellular tracers. When muscles are soaked in Ca 2+ -free solutions with 1 mM ECTA, the active zones become disorganized and isolated remnants of the double rows are found dissociated from the active zones. When venom was applied under these conditions dimples or protuberances still occurred mainly near the remnants of the double rows, even when these had migrated from their usual locations opposite the postjunctional folds. Thus, when transmitter release is stimulated and recylcing is vigorous, dimples or protuberances occur all over the presynaptic membrane whereas when transmitter release is stimulated, but recycling is impaired, as in venom treated terminals, dimples or protuberances occur only near the particles. These results show that quantal release of neurotransmitter occurs by exocytosis and that the sites of exocytosis are predetermined by the large intramembranous particles that normally line the edges of the active zone. On the other hand endocytosis of synaptic vesicles can occur either at the sites of exocytosis or nearby, at regions between the active zones. Supported by MDA Grant awarded to B. Ceccarelli.